Search Results (331)

Botulinum toxin (BoNT) causes flaccid paralysis by blocking the release of neurotransmitters. BoNTs associate with neurotoxin-associated proteins to form medium and large progenitor toxin complexes. The large progenitor toxin complex serotype A-62A (L-PTC/A-62A) specifically targets intestinal M cells for invasion, whereas large progenitor toxin complex serotype B-Okra (L-PTC/B-Okra) is mainly taken up by enterocytes and exhibits higher toxicity via the oral route. Hemagglutinin (HA) is a neurotoxin-associated protein that promotes BoNT absorption from the intestine and has carbohydrate-binding and barrier-disrupting activities. In this study, we established an in vitro reconstitution and purification system for recombinant L-PTC/B-Okra and created a recombinant L-PTC/B-Okra mutant rL-PTC/B-KA with carbohydrate-binding activity but not barrier-disrupting activity. rL-PTC/B-KA showed significantly reduced oral toxicity. Our results demonstrate that the B-Okra toxin disrupts the epithelial barrier of enterocytes and exerts oral toxicity. Full article

H5N1 Influenza A virus continues to pose a significant zoonotic threat, with increasing evidence of interspecies transmission and genetic adaptation. Previous studies primarily focused on avian or human isolates, with limited comprehensive analysis of H5N1 evolution across multiple mammalian hosts. Existing molecular surveillance often lags behind viral evolution; this study underscores the necessity for real-time monitoring of ongoing mutations affecting pathogenicity and transmissibility. Our goals are (1) to retrieve and analyze HA, NP and NA gene sequences of H5N1 Influenza A virus from diverse hosts, including humans, poultry and multiple mammalian species, to assess genetic diversity and evolutionary patterns and (2) to evaluate positive selection sites across the three major genes (HA, NP and NA) to determine adaptive mutations linked to host adaptation and viral survival. To achieve these goals, in this study, we considered (78 HA), (62 NP) and (61 NA) gene sequences from diverse hosts, including humans, poultry and multiple mammalian species, retrieved from the NCBI database. Phylogenetic analysis revealed distinct clade formations, indicating regional spread and cross-species transmission events, particularly from avian sources to mammals and humans. Selection pressure analysis identified positive selection across all three genes, suggesting adaptive mutations contributing to host adaptation and viral survival. Homology modeling and molecular dynamics simulations were performed to generate high-quality structural models of HA, NP and NA proteins, which were subsequently validated using multiple stereochemical parameters. Domain analysis confirmed conserved functional motifs, while protein–ligand docking demonstrated stable interactions at conserved binding sites, despite observed residue substitutions in recent isolates. Earlier research concentrated on HA alone; this study integrates HA, NP and NA genes for a broader understanding of viral evolution and adaptation. These findings highlight ongoing evolutionary changes in H5N1 genes that may enhance viral adaptability and pathogenicity, underscoring the need for continuous molecular surveillance and updated antiviral strategies. Full article

Influenza virus is a leading cause of global morbidity and mortality due to acute lower respiratory infection, even with the widespread use of multiple licensed influenza vaccines. However, antigenic drift during influenza replication can cause vaccine-induced antibodies to poorly neutralize influenza virus, thereby reducing vaccine effectiveness. To help overcome this problem, we leveraged a hydrogel platform with influenza hemagglutinin (HA) protein to induce prolonged antigen exposure. The hydrogel platform, Vaccine Self-Assembling Immune Matrix (VacSIM^®), in combination with recombinant influenza H1 or H3 HA protein antigens, increased antigen-specific antibody titers in vaccinated mice, which led to decreased disease severity after H1N1 infection for H1 HA-vaccinated mice and decreased lung viral titers after H3N2 challenge for H3 HA-vaccinated mice. Sera collected from mice immunized with VacSIM and HA also showed broader HAI activity, increasing by 1–3 log against a panel of influenza viruses. These results were consistent with the use of cocktail immunization, containing both an H1 and H3 HA, where mice immunized with VacSIM had an increase in antigen-specific antibody titers and decreased disease severity and lung viral titers against H1N1 and H3N2 influenza challenges, respectively. Finally, it was determined that a single immunization with VacSIM and H1 HA could provide protection against lethal H1N1 challenge compared to a group without VacSIM. In summary, we demonstrate that use of the slow-release platform VacSIM can improve the host immune response to vaccination and increase protection against influenza infection. Full article

Background/Objectives: Avian influenza represents a major threat to both animal and public health. Our group has tracked avian influenza viruses circulating in wild birds in Peru during the last 20 years. While most of these viruses are low-pathogenic avian influenza strains, some exhibit genetic changes that significantly diverge from common circulating viruses. We selected a highly divergent hemagglutinin H2 gene from a genetically characterized avian influenza virus to develop a recombinant protein using a baculovirus system. Methods: We administered 5 µg and 20 µg doses of the recombinant H2 protein (rH2) into 3-week-old chickens using an abdominal cavity inoculation model to evaluate the activation of innate immune responses. Chickens were euthanized at 24 and 72 h post inoculation and an abdominal lavage was performed to harvest the abdominal cavity content. Results: Infiltrating cells were counted and their cell viability was measured using an Annexin V/PI staining. At 24 h, a large proportion of infiltrating leukocytes were identified as heterophils, monocyte/macrophages and lymphocytes. These proportions changed at 72 h, with a decrease in heterophils and increase in monocyte and lymphocyte pools. We observed strong cellular activity in abdominal leukocytes at 24 h, with a decline in activation levels at 72 h. Cytokine expression suggested a tightly regulated immune response during the 72 h period, while a more sustained response was observed at the 20 µg dose. Antibody levels demonstrated the capacity of the rH2 protein to induce long-term responses. Conclusions: These results revealed that the baculovirus-expressed rH2 protein induces a controlled immune activation, a long-term immune response, holding promise as a potential vaccine candidate for animal health. Full article

Canine distemper virus (CDV) is a multi-host morbillivirus whose evolution and host-switching capacity are largely determined by its hemagglutinin (H) gene. To reconsider the molecular evolution of this critical gene, we performed comprehensive phylogenetic, selection, and structural analyses on a curated dataset of 68 representative global H gene sequences. Our phylogenetic reconstruction confirmed the segregation of sequences into distinct, geographically associated lineages. To provide stronger evidence for viral adaptation, we performed a site-specific selection analysis, which identified 15 amino acid sites in the H protein undergoing significant episodic positive selection. Crucially, the majority of the known SLAM and Nectin-4 receptor-binding residues were found to be among these positively selected sites. We further contextualized these findings by mapping the sites onto a 3D homology model of the H protein, which confirmed their location on the exposed surfaces of the receptor-binding domain. This compilation provides quantitative evidence that the key functional regions of the H protein are direct targets for adaptive evolution, which has significant implications for understanding host tropism and the ongoing challenge of vaccine mismatch. Full article

Influenza B viruses, divided into B/Victoria and B/Yamagata lineages, have not had B/Yamagata isolates after 2020. A study evaluated immunity to influenza B surface antigens hemagglutinin (HA) and neuraminidase (NA) in 138 patient sera from 2023 and 23 pairs of sera from 2018 to 2019 vaccine recipients. The phylogenetic tree of the influenza B virus, based on HA and NA genes, shows that the Yamagata lineage evolves gradually, while the Victoria lineage exhibits rapid mutations with short branches. In 2023, mean levels of antibodies to HA and NA of B/Yamagata virus were higher than to B/Victoria, despite no cases of B/Yamagata lineage isolation after 2020. The titers of antibodies to NA of B/Yamagata statistically significantly differed among individuals born before and after 1988. Among patients examined in 2018–2019, neuraminidase-inhibiting (NI) antibody titers before vaccination were higher to B/Yamagata than to B/Victoria, and NI antibodies to B/Victoria and B/Yamagata positively correlated with neutralizing antibodies to B/Victoria virus before and after vaccination. Immunity to B/Yamagata virus was stronger in 2023, despite no isolation since 2020, probably due to the presence of cross-reactive antibodies from B/Victoria infections or vaccinations. Antibodies to NA of B/Victoria and B/Yamagata in 2023 correlated significantly in patients born before 1988, potentially supporting the concept of ‘antigenic sin’ phenomenon for influenza B viruses. The fact that NI antibody titers to B/Victoria and B/Yamagata correlated with neutralizing antibody titers to B/Victoria may suggest broad cross-protection. Studying influenza B virus NA antigenic properties helps understand the evolution and antigenic competition of HA and NA. Full article

Background: During infection with the cytomegalovirus (CMV), the membrane system of the infected cell is remodelled into a megastructure called the assembly compartment (AC). These extensive changes may involve the manipulation of the host cell proteome by targeting a pleiotropic function of the cell such as ubiquitination (Ub). In this study, we investigate whether the Ub system is required for the establishment and maintenance of the AC in murine CMV (MCMV)-infected cells Methods: NIH3T3 cells were infected with wild-type and recombinant MCMVs and the Ub system was inhibited with PYR-41. The expression of viral and host cell proteins was analyzed by Western blot. AC formation was monitored by immunofluorescence with confocal imaging and long-term live imaging as the dislocation of the Golgi and expansion of Rab10-positive tubular membranes (Rab10 TMs). A cell line with inducible expression of hemagglutinin (HA)-Ub was constructed to monitor ubiquitination. siRNA was used to deplete host cell factors. Infectious virion production was monitored using the plaque assay. Results: The Ub system is required for the establishment of the infection, progression of the replication cycle, viral gene expression and production of infectious virions. The Ub system also regulates the establishment and maintenance of the AC, including the expansion of Rab10 TMs. Increased ubiquitination of WASHC1, which is recruited to the machinery that drives the growth of Rab10 TMs, is consistent with Ub-dependent rheostatic control of membrane tubulation and the continued expansion of Rab10 TMs. Conclusions: The Ub system is intensively utilized at all stages of the MCMV replication cycle, including the reorganization of the membrane system into the AC. Disruption of rheostatic control of the membrane tubulation by ubiquitination and expansion of Rab10 TREs within the AC may contribute to the development of a sufficient amount of tubular membranes for virion envelopment. Full article

Background: Influenza A viruses (IAV) cause seasonal flu and occasional pandemics. In addition, the potential for the emergence of new strains presents unknown challenges for public health. Face masks and other personal protective equipment (PPE) can act as barriers that prevent the spread of these viruses. Metal ions embedded into PPE have been demonstrated to inactivate respiratory viruses, but the underlying mechanism of inactivation and potential for resistance is presently not well understood. Methods: In this study, we used hemagglutination assays to quantify the effect of zinc ions on IAV sialic acid receptor binding. We varied the zinc concentration, incubation time, incubation temperature, and passaged IAV in the presence of zinc ions to investigate if resistance to zinc ions could evolve. Results: We found that zinc ions impact the ability of IAV particles to hemagglutinate and observed inhibition within 1 min of exposure. Maximum inhibition was achieved within 1 h and sustained for at least 24 h in a concentration-dependent manner. Inhibition was also temperature-dependent, and optimal above room temperature. Serial passaging of IAV in the presence of zinc ions did not result in resistance. Conclusions: e conclude that zinc ions prevent IAV hemagglutination in a concentration and temperature-dependent manner for at least 24 h. Overall, these findings are in line with previous observations indicating that zinc-embedded materials can inactivate the IAV hemagglutinin and SARS-CoV-2 spike proteins, and they support work toward developing robust, passive, self-cleaning antiviral barriers in PPE. Full article

Background: Fowl typhoid (FT), a septicemic infection caused by Salmonella Gallinarum (SG), and H9N2 avian influenza are two economically important diseases that significantly affect the global poultry industry. Methods: We exploited the live attenuated Salmonella Gallinarum (SG) mutant JOL3062 (SG: ∆lon ∆pagL ∆asd) as a delivery system for H9N2 antigens to induce an immunoprotective response against both H9N2 and FT. To enhance immune protection against H9N2, a prokaryotic and eukaryotic dual expression plasmid, pJHL270, was employed. The hemagglutinin (HA) consensus sequence from South Korean avian influenza A virus (AIV) was cloned under the Ptrc promoter for prokaryotic expression, and the B cell epitope of neuraminidase (NA) linked with matrix protein 2 (M2e) was placed for eukaryotic expression. In vitro and in vivo expressions of the H9N2 antigens were validated by qRT-PCR and Western blot, respectively. Results: Oral immunization with JOL3121 induced a significant increase in SG and H9N2-specific serum IgY and cloacal swab IgA antibodies, confirming humoral and mucosal immune responses. Furthermore, FACS analysis showed increased CD4+ and CD8+ T cell populations. On day 28 post-immunization, there was a substantial rise in the hemagglutination inhibition titer in the immunized birds, demonstrating neutralization capabilities of immunization. Both IFN-γ and IL-4 demonstrated a significant increase, indicating a balance of Th1 and Th2 responses. Intranasal challenge with the H9N2 Y280 strain resulted in minimal to no clinical signs with significantly lower lung viral titer in the JOL3121 group. Upon SG wildtype challenge, the immunized birds in the JOL3121 group yielded 20% mortality, while 80% mortality was recorded in the PBS control group. Additionally, bacterial load in the spleen and liver was significantly lower in the immunized birds. Conclusions: The current vaccine model, designed with a host-specific pathogen, SG, delivers a robust immune boost that could enhance dual protection against FT and H9N2 infection, both being significant diseases in poultry, as well as ensure public health. Full article

A novel bivalent oral vaccine candidate against H5N1 and H9N2 avian influenza virus (AIV) was developed using Lactobacillus surface display technology without genetic modification. The hemagglutinin subunit 1 (HA1) antigens from both subtypes were fused to the surface layer-binding domain of Lactobacillus crispatus K313, expressed in Escherichia coli, and purified. Wild-type Lactobacillus johnsonii H31, isolated from chicken intestine, served as a delivery vehicle by adsorbing and stably displaying the HA1 proteins on its surface. This approach eliminates the need for bacterial engineering while utilizing lactobacilli’s natural capacity to protect surface-displayed antigens, as evidenced by HA1’s protease resistance. Mouse immunization studies demonstrated induction of strong systemic IgG and mucosal IgA responses against both H5N1 and H9N2 HA1. The system offers several advantages, including safety through non-GMO probiotics, potential for multivalent vaccine expansion, and intrinsic antigen protection by lactobacilli. These findings suggest this platform could enable development of cost-effective, multivalent AIV vaccines. Full article

(1) Background: The highly pathogenic avian influenza virus H5N1 clade 2.3.4.4b is an emerging threat that poses a great risk to the poultry industry. A few human cases have been linked to the infection with this clade in many parts of the world, including the USA. Unfortunately, there are no specific vaccines or antiviral drugs that could help prevent and treat the infection caused by this virus in birds. Our major objective is to identify/repurpose some (novel/known) antiviral compounds that may inhibit viral replication by targeting some key viral proteins. (2) Methods: We used state-of-the-art machine learning tools such as molecular docking and MD-simulation methods from Biovia Discovery Studio (v24.1.0.321712). The key target proteins such as hemagglutinin (HA), neuraminidase (NA), Matrix-2 protein (M2), and the cap-binding domain of PB2 (PB2/CBD) homology models were validated through structural assessment via DOPE scores, Ramachandran plots, and Verify-3D metrics, ensuring reliable structural representations, confirming their reliability for subsequent in silico approaches. These approaches include molecular docking followed by molecular dynamics simulation for 50 nanoseconds (ns), highlighting the structural stability and compactness of the docked complexes. (3) Results: Molecular docking revealed strong binding affinities for both sofosbuvir and GS441524, particularly with the NA and PB2/CBD protein targets. Among them, GS441524 exhibited superior interaction scores and a greater number of hydrogen bonds with key functional residues of NA and PB2/CBD. The MM-GBSA binding free energy calculations further supported these findings, as GS441524 displayed more favorable binding energies compared to several known standard inhibitors, including F0045S for HA, Zanamivir for NA, Rimantadine and Amantadine for M2, and PB2-39 for PB2/CBD. Additionally, 50 ns molecular dynamics simulations highlighted the structural stability and compactness of the GS441524-PB2/CBD complex, further supporting its potential as a promising antiviral candidate. Furthermore, hydrogen bond monitor analysis over the 50 ns simulation confirmed persistent and specific interactions between the ligand and proteins, suggesting that GS441524 may effectively inhibit the NA, and PB2/CBD might potentially disrupt PB2-mediated RNA synthesis. (4) Conclusions: Our findings are consistent with previous evidence supporting the antiviral activity of certain nucleoside analog inhibitors, including GS441524, against various coronaviruses. These results further support the potential repurposing of GS441524 as a promising therapeutic candidate against H5N1 avian influenza clade 2.3.4.4b. However, further functional studies are required to validate these in silico predictions and support the inhibitory action of GS441524 against the targeted proteins of H5N1, specifically clade 2.3.4.4b. Full article

The global burden of respiratory viral infections is notable, which is attributed to their higher transmissibility compared to other viral diseases. Respiratory viruses are seen to have evolved resistance to available treatment options. Although vaccines and antiviral drugs control some respiratory viruses, this control is limited due to unexpected events, such as mutations and the development of antiviral resistance. The technology of proteolysis-targeting chimeras (PROTACs) has been emerging as a novel technology in viral therapeutics. These are small molecules that can selectively degrade target proteins via the ubiquitin–proteasome pathway. PROTACs as a therapy were initially developed against cancer, but they have recently shown promising results in their antiviral mechanisms by targeting viral and/or host proteins involved in the pathogenesis of viral infections. In this review, we elaborate on the antiviral potential of PROTACs as therapeutic agents and their potential as vaccine components against important respiratory viral pathogens, including influenza viruses, coronaviruses (SARS-CoV-2), and respiratory syncytial virus. Advanced applications of PROTAC antiviral strategies, such as hemagglutinin and neuraminidase degraders for influenza and spike proteins of SARS-CoV-2, are detailed in this review. Additionally, the role of PROTACs in targeting cellular mechanisms within the host, thereby preventing viral pathogenesis and eliciting an antiviral effect, is discussed. The potential of PROTACs as vaccines, utilizing proteasome-based virus attenuation to achieve a robust protective immune response, while ensuring safety and enhancing efficient production, is also presented. With the promises exhibited by PROTACs, this technology faces significant challenges, including the emergence of novel viral strains, tissue-specific expression of E3 ligases, and pharmacokinetic constraints. With advanced computational design in molecular platforms, PROTAC-based antiviral development offers an alternative, transformative path in tackling respiratory viruses. Full article

Influenza A virus (IAV) poses significant public health challenges due to its rapid mutation and drug resistance, necessitating novel antiviral strategies. Rhododendron brachycarpum, traditionally employed in folk medicine to treat inflammatory conditions, contains bioactive flavonoids with potential antiviral effects. In this study, we investigated the anti-influenza activity of R. brachycarpum leaf extract and identified hyperoside (quercetin-3-O-galactoside) as the active constituent. The crude extract and its n-butanol fraction markedly reduced IAV replication in Madin–Darby canine kidney (MDCK) cells, with IC50/CC50 values of 74.51/201.09 μg/mL and 24.5/113.1 μg/mL, respectively. Hyperoside, purified via bioactivity-guided fractionation and HPLC analysis, demonstrated potent antiviral activity, with an IC50 of 66.59 μM (30.92 μg/mL) and a CC50 of 318.9 μM (148.1 μg/mL), indicating a favorable selectivity index. It significantly suppressed viral mRNA and protein expression in infected cells. Time-of-addition and hemagglutination inhibition assays suggested that hyperoside exerts antiviral effects during early infection stages, likely interfering with viral entry. In silico molecular docking analysis further supported this mechanism, revealing that hyperoside binds strongly to the receptor-binding domain of hemagglutinin (−11.5 kcal/mol), potentially blocking viral attachment. These findings reveal that hyperoside is a major antiviral component of R. brachycarpum and underscore its therapeutic potential as a natural antiviral candidate against IAV infections. Full article

Background/Objectives: Developing next-generation mRNA-based seasonal influenza vaccines remains challenging, primarily because of the relatively low immunogenicity of influenza B hemagglutinin (HA) antigens. We describe a systematic vaccine development strategy that combined vector and antigen design optimization. Methods: Novel untranslated region (UTR) sequences and a hybrid poly(A) tail were used to increase plasmid stability and mRNA expression. Fusion proteins containing HA antigens linked by T4 foldon domains were engineered to enhance the immune responses against influenza B HA antigens and to permit the expression of multiple HA ectodomains from a single mRNA species. The vaccine performance was verified in a traditional encapsulated lipid nanoparticle (LNP) formulation that requires long-term storage at temperatures below −15 °C as well as in a proprietary thermo-stable LNP formulation developed for the long-term storage of the mRNA vaccine at 2–8 °C. Results: In preclinical studies, our next-generation seasonal influenza vaccine tested alone or as a combination influenza/COVID-19 mRNA vaccine elicited hemagglutination inhibition (HAI) titers significantly higher than Fluzone HD, a commercial inactivated influenza vaccine, across all 2024/2025 seasonal influenza strains, including the B/Victoria lineage strain. At the same time, the combination mRNA vaccine demonstrated superior neutralizing antibody titers to 2023/2024 Spikevax, a commercial COVID-19 comparator mRNA vaccine. Conclusions: Our data demonstrate that the multimerization of antigens expressed as complex fusion proteins is a powerful antigen design approach that may be broadly applied toward mRNA vaccine development. Full article

Background: canine distemper (CD) is a highly contagious and fatal disease caused by canine distemper virus (CDV), posing a significant threat to carnivores. New CDV strain circulation and multi-species infection may lead to the potential dilemma of safety concern and insufficient efficacy of the commercial modified live vaccines. Safe and effective vaccines for canine and wildlife prevention of CD need to be continuously updated and developed. Methods: we developed two DNA vaccines, p17F-LNP and p17H-LNP, encoding the fusion protein (F) or hemagglutinin protein (H) of a field CDV strain (HLJ17) and encapsulated in lipid nanoparticles (LNPs). Serum neutralizing antibody (NAb) was evaluated via neutralization tests, and mouse serum cytokine detection were evaluated via ELISA. Results: immunization of p17F-LNP and p17H-LNP monovalent or bivalent were safe, and induced robust CDV NAb and cytokine responses in mice. LNP encapsulation improved immune responses compared to naked DNA formulation, and the bivalent formulation of p17F-LNP and p17H-LNP (p17F/H-LNP) exhibited synergistic effects with a high level of immune responses. Moreover, two doses of p17F/H-LNP induced long-lasting CDV NAb for over 300 days in dogs, and prime and boost NAb responses in foxes and raccoon dogs. Conclusions: the preliminary findings provided here warrant further development of the p17F/H-LNP vaccine for animal targets against CDV infection. Full article